Malaria remains the most important parasitic disease in the world, affecting hundreds of millions of people and killing almost a million every year. Antimalarial drugs are the mainstay of malaria control, but the spread of parasite resistance to most antimalarials is of grave concern. To address this concern, concerted efforts have been directed at discovering and developing new antimalarial drugs, with some encouraging early results. Yet, it is clear that for the foreseeable future we would need to feed the pipeline of antimalarials, because resistance to new drugs is sure to arise. Recently, we have discovered a series of compounds with a pyrazole core that demonstrate highly potent antimalarial activity. These compounds are undergoing the process of development as an antimalarial drug with a novel structure. Our data also suggest that their mode of action is likely to target a vital and a hitherto unknown vulnerable pathway in malaria parasites. By applying a variety of approaches, we propose to uncover the nature of this pathway and its molecular components. The aims we propose here will use both empirical and hypothesis-driven approaches to identify molecular mechanisms underlying this promising series of compounds. We will employ a variety of methods to assess changes occurring in P. falciparum blood stages as they are rapidly being killed by the pyrazole compounds. These will include: phosphoprotein profiling, transcriptome changes, changes in metabolites, calcium homeostasis, and morphological changes at both light and electron microscopic levels. These studies will provide clues, as well as confirmation, regarding the pathways affected by the compounds. Recent studies suggest that pyrazoles and another series of potent antimalarials, spiroindolones, may be working through a common pathway. We will assess common features in mode of action of these two chemically distinct compounds. We will test a hypothesis that the pyrazole compounds are targeting a pathway similar to phosphorylation regulatory mechanisms observed in plants. This hypothesis is based on our initial observation of mutations discovered through whole genome sequencing of resistant parasites that we found to be important in imparting the resistance phenotype. Overall, these studies has the potential not only to understand mechanism of action for promising antimalarials but also to reveal new targets for future investigations.) PUBLIC HEALTH RELEVANCE: There is an urgent need to discover, develop and deploy new antimalarial drugs. This project is based on recent discovery of highly potent antimalarial compounds with a novel chemotype. The project aims to uncover a vulnerable molecular pathway affected by these compounds and to identify additional potential targets within the pathway. Successful identification of the target will also permit better design of potential drugs.